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  • Title: Efficient in vivo knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA.
    Author: Krom YD, Fallaux FJ, Que I, Lowik C, van Dijk KW.
    Journal: BMC Biotechnol; 2006 Feb 28; 6():11. PubMed ID: 16507095.
    Abstract:
    BACKGROUND: Adenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor alpha (ERalpha). RESULTS: Two different shRNA constructs derived from the murine ERalpha coding sequence were designed (shERalpha). In vitro, transfection of three mouse cell lines with pSUPER-shERalpha constructs resulted in up to 80% reduction of endogenous ERalpha activity. A single mismatch in the target sequence eliminated the reduction of ERalpha activity, demonstrating the specificity of shERalpha. The subsequently generated Ad.shERalpha vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERalpha resulted in 70% reduced hepatic mouse ERalpha mRNA levels. Co-injection of Ad.shERalpha with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection. CONCLUSION: Ad mediated transfer of shERalpha expression constructs results in efficient and specific knockdown of endogenous ERalpha transcription both in vitro and in vivo.
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